The use of combinatorial approaches for protein identification, characterization and modification has been highly successful in both academic and commercial research and development. In this respect, filamentous bacteriophage, or phage, display technology has paved the way being the first library platform and still thrones as the dominating technology. Thus, phage display is widely applied in both basic and applied protein discovery, as well as in development of both novel protein-based diagnostics and therapeutic, which are the class of compounds most rapidly growing world-wide.
The principle of combinatorial phage display technology is based on the genotype-phenotype linkage offered by the property that each virion will only display on its surface the very same proteins that are encoded by the genome encapsulated by its protein coat. The phage particle itself is highly resistant to a variety of physiochemical conditions; hence phage display offers superior versatility in many selection regimes as compared to competing combinatorial technologies.
Phage display of heterologous polypeptides has been achieved using all five structural proteins of the filamentous phage coat, but only pIII- and to some extent pVIII-display have gained widespread use (FIG. 1).
When the heterologous fusion is only a short peptide, multivalent display systems using phage genome-based vectors are preferred, whereas for larger fusions requiring folded domains most applications will benefit from phagemid systems. In the latter case, antibody-pIII phage display is by far dominating the field, but alternative scaffolds are emerging at dawns early light, continuing the need for expansion of protein engineering tools of tomorrow. A highly desired application is to effectively obtain high affinity specific peptide or protein binders from a phage display library simply by infecting bacteria with phage virions while still bound to its target.
Endemann and Model, 1995 (PMID: 7616570), reported that the minor coat pIX was not functional with another protein fused to its N-terminus. Therefore, this report concluded that pIX cannot be used for phage display.
Both Gao et al. (PMID: 10339535, 12239343 and WO0071694) and Khalil et al. (PMID: 17360403) have later shown that N-terminal pIX fusion display is allowed when expressed from phagemids and used in combination with signal sequence-dependent periplasmic targeting. In these systems, complementation takes place as wt pIX is donated from the helper phage genome upon phagemid rescue.
The phagemid as disclosed in FIG. 2A of WO0071694 (without fusion protein inserted) and FIG. 2B of WO0071694 (with fusion protein inserted) clearly comprises a pelB signal sequence (with figure text on page 7, lines 2-14).
As mentioned above, it had previously been suggested that pIX was not functional with another protein fused to the N termini, and Gao et. al. gave two possible reasons for their success, either alone or by the combination of both.
One possible reason was that a prokaryotic leader sequence (signal sequence) was attached N-terminally to the fusion proteins, thus ensuring targeting of the recombinant protein to the periplasmic space and thereby prevented accumulation in the cytoplasm.
Another possible reason was that the recombinant proteins were expressed from a phagemid, not a phage genome as by Endemann and Model, hence wild type pIX from the helper phage inevitably needed for phagemid rescue were complementing the recombinant pIX fusion proteins, thus preserving wild type functionality that otherwise may have been lost due to the recombinant modification. I.e. the phages would comprise a mix of wild-type and fusion proteins.
Khalil et al. (PMID: 17360403) describes an application exploiting the feature of a bispecific filamentous phage virion in which an exogenous peptide is displayed at each distal tip of the very same virion. They achieved this by using the combination of a common pIII phage genome vector complementing a prokaryotic signal sequence dependent pIX display phagemid. In this setting, the phage genome vector served as a helper phage in rescuing the phagemid.